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PurposeProspective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade.Patients and methodsWe performed a multicenter phase II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink.ResultsEighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1-7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3-4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3-5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8+ and CD8+/PD-1+/Ki-67+ T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood.ConclusionsWe demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies.

BACKGROUND. Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse. METHODS. We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. Highresolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion. RESULTS. In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. CONCLUSION. Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies. TRIAL REGISTRATION. ClinicalTrials.gov NCT04024761. FUNDING. Dunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society., Introduction Relapse of acute myeloid leukemia (AML) occurs in up to 50% of patients after allogeneic hematopoietic stem cell transplant (HCT) and heralds an extremely poor prognosis (1). In several [...]

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti–programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting. Editor's summary: Immune checkpoint blockade (ICB) such as anti-PD-1 reinvigorates tumor-specific T cell responses, but the mechanisms underlying specific clinical responses remain unclear. Using single-cell transcriptomics and TCR sequencing, Oliveira et al. analyzed tumor specimens from patients with head and neck cancer enrolled in a phase II clinical trial testing two doses of neoadjuvant anti–PD-1 before surgical resection. Tumors responding to anti–PD-1 contained a baseline population of ZNF683+CD8+ T cells expressing genes associated with T cell exhaustion, tissue resident memory, and cytotoxicity. In paired pre- and post-treatment biopsies, ZNF683+CD8+ T cells were clonally expanded and exhibited the strongest change in patients responding to ICB. These findings identify reinvigoration of cytotoxicity among ZNF683+CD8+ T cells as a likely mechanism underlying response to neoadjuvant anti–PD-1 in head and neck cancer. —Claire Olingy [ABSTRACT FROM AUTHOR]

Simultaneous disruption of two different protein-protein interactions within the (APC/C-Cdc20)-substrate complex can synergistically inhibit APC/C-dependent proteolysis and mitotic exit. APC/C activation blocks cell proliferation The anaphase-promoting complex/cyclosome (APC/C) is a thirteen-subunit ubiquitin ligase enzyme that mediates progression through mitotic cell division by targeting substrate proteins for degradation by the ubiquitin-proteasome system. During mitosis, the protein Cdc20 binds to APC/C to activate it, and the two proteins together recognize target substrates. Blocking mitotic exit by blocking APC/C activity could induce tumour-cell death and therefore might be of potential therapeutic value. This study shows that simultaneous disruption of two different protein-protein interactions within the (APC/C-Cdc20)-substrate complex can synergistically inhibit APC/C-dependent proteolysis and mitotic exit. Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome.sup.1,2. Because blocking mitotic exit is an effective approach for inducing tumour cell death.sup.3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box).sup.6,7,8,9,10,11,12,13,14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C-Cdc20 interaction.sup.15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine., Author(s): Katharine L. Sackton [sup.1] , Nevena Dimova [sup.1] , Xing Zeng [sup.1] , Wei Tian [sup.2] [sup.6] , Mengmeng Zhang [sup.1] , Timothy B. Sackton [sup.3] , Johnathan Meaders [...]